Pyruvate is the key glycolytic intermediate of all mammalian cells. As discussed in more detail below, this substance and pharmaceutically acceptable derivatives thereof are useful as biological stimulating agents.
1) Pyruvate compartmentalization and cytoplasmic phophorylation potential: Intracellular pyruvate is usually derived from glucose, i.e. it is a key glycolytic intermediate of all mammalian cells. It can also be formed from extracellular lactate via the lactate dehydrogenase reaction, In situations where pyruvate is employed as an exogneous metabolic substrate, i.e. where its extracellular concentration is sufficiently raised, pyruvate functions as a precursor of lactate by reversing the lactate dehydrogenase reaction. Further, in contrast to alternative metabolic fuels such as acetate and also lactate, pyruvate has recently been established by applicant as an agent that consistently improves key indices of the cytoplasmic phophorylation potential of creatine phosphate (ratios of the concentrations of creatine phosphate (CrP) to inorganic phosphate (P.sub.1), to that of creatine (Cr), or to the product of the concentrations of creatine and inorganic phosphate, [CrP]/([Cr]*[Pi]); a formally similar concentration ratio is the phosphorylation potential of ATP, [ATP]/([ADP]*[P.sub.1 ]), which is coupled to and in most cases in equilibrium with [CrP]/([Cr]*[C.sub.1 ]), an effect mediated by the powerful magnesium- and pH- dependent enzyme creatine kinase; this enzyme is present in high concentrations in striated and smooth muscle (heart, vascular smooth muscle, skeletal muscle) and brain, but not in liver and kidney. [ATP]/([ADP]*[P.sub.1 ]) is a major determinant of the actual free energy available from cellular ATP hydrolysis according to the following equation: EQU G.sub.ATP = G.sup.0 .sub.ATP +R.multidot.T.multidot.in([ADP].multidot.[P.sub.1 /[ATP])
in which G.sup.0.sub.ATP is the (relatively constant) standard free energy change of ATP hydrolysis under conditions prevailing in vivo (-32.35 kJ/mol, pH 7.2, free cytoplasmic magnesium concentration&lt;1 mM), R=gas constant (8.314 J/K*mol) and T=absolute temperature in degrees Kelvin (K). Thus, during alterations of physiologic states and under many pathophysiological states investigated so far, [ATP]/([ADP]*[P.sub.1 ]) can change considerably, whereas the G.sup.0.sub.ATP -term changes relatively little.
Pyruvate, administered in doses between 2 to 10 mM, has recently been demonstrated by applicant to raise the phosphorylation potential in a dose-dependent manner in normal, but especially in reversibly damaged (ischemia/reperfusion protocols) heart models of guinea pig, dog and pig. Thus, pyruvate administration can somewhat (by about 4 to 6%) improve the free energy available for cellular phosphorylations and energy consuming ion transporters as well.
Pyruvate is centered at the compartmental interface between cytoplasma and mitochondria; applicant has recently shown that it is linked via the cytoplasmic NAD*/NADH system (which is under the join control of two major cytoplasmic dehydrogenases, the lactate dehydrogenase and the glyceraldehyde-3-phosphate dehydrogenase) to the cytoplasmic phosphorylation potential. Thus, pyruvate is coupled to [ATP]/([ADP]*[P.sub.1 ]) in its capacity as substrate of lactate dehydrogenase, which can affect the NAD.sup.+ /NADH system which in turn is stoichiometrically coupled the combined glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction; the latter enzyme system involves ATP, ADP, and P.sub.1 as reactants, i.e. is linked directly to the cytoplasmic [ATP]/([ADP]*[P.sub.1 ]) rather than the CrP phosphorylation potential, [CrP]/([Cr]*[P.sub.1 ]), but applicant has demonstrated that it also can be estimated using the reactants of the glyceraldehyde-3-phosphate dehydrogenase combined with those of the lactate dehydrogenase.
2) Pyruvate dehydrogenase: Pyruvate is also the immediate substrate of the powerful mitochondrial pyruvate dehydrogenase enzyme complex (PDH), the main mechanism that controls entry of carbohydrate and lactate carbon into the citric acid cycle for end-oxidation (formation of water and carbon dioxide) coupled with oxidative phosphorylation (formation of ATP from ADP and inorganic phosphate). In addition, pyruvate, not lactate or acetate, is auto-catalytically active at the PDH enzyme complex; thus pyruvate stimulates covalent modification (dephosphorylation) of the interconvertible PDH complex, which results in increased activity of the PDH; this in turn stimulates oxidative decarboxylation of pyruvate to acetyl-CoA and carbon dioxide and hence facilitates complete conversion of cellular glucose- and lactate-carbon to water and carbon dioxide (see below). The net effect of these changes is an increase availability of NADH in the mitochondria, thereby improving the ability of the cell to adapt promptly to changing energy demands.
3) Pyruvate Carboxylase: Another important feature of pyruvate only (not of other substrates such as lactate or acetate), is that it functions as the immediate substrate of the CO.sub.2 -fixing-enzyme pyruvate carboxylase. This enzyme is present in relatively small amounts in liver and heart and probably other organs as well; but it is important, since it assimilates metabolic CO.sub.2 by adding it to the carbon-3-skeleton of pyruvate, thus providing the mammalian cell with an "anaplerotic" mechanism; the overall effect is the netsynthesis of mitochondrial carbon-4-skeletons, which helps to adequately maintain the concentration of the rather small but absolutely vital carbon-4-oxaloacetate pool in the mitochondria. Oxaloacetate is crucial for the mitochondrial condensing enzyme (citrate synthase) which catalyzes the aldol condensation between the methyl group of acetyl-CoA (generated in the PDH reaction or derived from ketone body or fatty acid metabolic pathways) and the carbonyl group of oxaloacetate resulting in the formation of citrate and coenzyme A. Oxaloacetate, an alpha-ketoacid like pyruvate and alpha-ketoglutarate, is normally present in the mitochondrial matrix in only very small concentrations; it is clear therefore that maintenance of its mitochondrial concentration can become crucial for adequate citric acid cycle turnover, the mechanism which provides the necessary reducing equivalents (NADH.sub.2, FADH.sub.2) for the respiratory chain which in turn ensures maintenance of ATP synthesis (oxidative phosphorylation) and hence cellular energy status (phosphorylation potential).
4) Lactate dehydrogenase and cytoplasmic NADH.sub.2 : At physiological pH of 7.0 to 7.4 pyruvic acid, because of its relatively low pK value of 2.49, is virtually completely dissociated into the negatively charged pyruvate anion and the positively charged H.sup.+ cation. It is known that the pyruvate anion (but probably also the undissociated free pyruvic acid), if administered in sufficient quantities, lowers the cytoplasmic [NADH]*[H.sup.+ ]/[NAD.sup.+ ] ratio in cellular systems that contain lactate dehydrogenase. This effect is often referred to as the oxidizing effect of pyruvate. It has been recently demonstrated by applicant that this effect of pyruvate can prevent the normal accumulation of cytoplasmic NADH.sub.2 during experimental cardiac ischemia [1]. This special oxidizing mechanism of pyruvate is potentially of great clinical significance, as extramitochondrial NADH.sub.2 has been found by others to be hazardous for isolated heart mitochondria (not for isolated liver mitochondria); in isolated heart mitochondria NADH.sub.2 lowers respiratory control and impairs energy coupling (ATP/oxygen ratio decreases) during readmission of oxygen; simultaneously the anoxic/reoxygenated NADH.sub. 2 -exposed cardiac mitochondria begin to double the generation of the pathological superoxide anion (O.sub.2 -) [2]; the superoxide anion belongs to a special class of reactive oxygen species usually called oxygen-derived free radicals; these molecular species have been implicated in the pathogenesis of oxidative stress during ischemia/reperfusion conditions of several organs such as heart, kidney and liver [3-6]. Both the impairment of energy coupling and the pathological superoxide anion formation were only observed when the isolated heart mitochondria were incubated with NADH.sub.2, not during acidosis or anoxia per se, i.e. in the absence of NADH.sub.2. Considering these new findings from isolated cardiac mitochondria, the recent demonstration by applicant that pyruvate can prevent large accumulations of NADH.sub.2 during ischemia [1], it becomes evident that pyruvate- via its oxydizing effect per se--could be instrumental could be instrumental in maintaining adequate mitochondrial energy coupling during subsequent reperfusion. According to the above rationale this would additionally be associated with a lower production rate of potentially dangerous oxygen-derived free radicals (superoxide anions). It is readily seen that these redox-effects of pyruvate may lead to improved reperfusion energetics and hence functional recovery in any organ that contains sufficient amounts of lactate dehydrogenase. As for the heart, anticipated improvements include reduced myocardial stunning, reduced probability of arrhythmias and ventricular fibrillation, reduced accumulation of sodium and hence calcium; attenuation of cytoplasmic calcium accumulation is particularly important, since unphysiologically high concentrations of the calcium ion can activate dangerous autolytic enzymes such as phospholipases (membrane lipid damage), proteases (cytoskeletal protein and enzyme damage), and endonucleases (DNA strand breaks [7]).
5) Fenton reaction, Fe.sup.2+ (ferrous Ion) and reducing equivalents: There is yet another reason why the cytoplasmic redox effects of high concentrations of pyruvate could be beneficial during ischemia-acidosis/reperfusion situations; this mechanism concerns the formation of cytotoxic oxygen-derived free radicals via Fenton-type reactions. This is the case because the Fenton reaction is the mechanism responsible for generation of the particularly cytotoxic hydroxyl radical; the reaction requires catalytic amounts of free Fe.sup.2+ (ferrous Ion) which interacts with hydrogen peroxide or the superoxide anion to yield three products: Fe.sup.3+ plus hydroxyl ion (both relatively inert) and the extremely reactive and thereof recytotoxic hydroxyl radical [8]. Normally most of cellular iron is complexed in the form of Fe.sup.3+ by ferritin or stored as haem-type-iron in proteins, enzymes, and cofactors. These complexes usually contain iron but are not themselves substrates or catalysts in Fenton-type reactions. However, during accumulation of reducing equivalents (FADH.sub.2 FMN.sub.2, NADH.sub.2) in ischemic/anoxic/infarcted organs, reduced flavins can bring about the reductive release of iron from ferritin [9]; this raises the concentration of free Fe2+ (ferrous Ion) which may then be available to react with hydrogen peroxide/superoxide anion of, e.g., mitochondrial origin during subsequent reperfusion. Indeed, during rat cardiac ischemia, accumulation of free Fe.sup.2+ (ferrous Ion) has recently been demonstrated, especially when ischemia was associated with significant cellular acidosis [10]. Since pyruvate treatment can help to metabolically neutralize cellular H.sup.+ (as opposed to direct chemical buffering by, e.g., bicarbonate) and also has been shown by applicant to prevent accumulation of reducing equivalents during ischemia by "clamping" the cytoplasmic redox status at the normoxic level [1], it is easily seen that acute parenteral pyruvate has the potential to attenuate or perhaps even completely prevent the intracellular reductive liberation of free Fe.sup.2+ (ferrous ion) during acidotic ischemia/anoxia. Applicant proposes that this special redox-affect of pyruvate Has the potential to attenuate/prevent Intracellular Fenton-type reactions, which in turn would diminish or perhaps even completely eliminate the formation of the cytotoxic hydroxyl radical from hydrogen peroxide or superoxide anion. Consequently, oxidative stress due to reperfusion/reoxygenation can be expected to be minimized, if adequate pyruvate therapy can be timely implemented to minimize the accumulation of reducing equivalents during pre-reperfusion ischemia/anoxia conditions. This particular and novel pyruvate mechanism will likely apply to all mammalian organs that contain lactate dehydrogenase and ferritin iron (e.g., brain, heart, kidney, lung); probable exceptions are liver and spleen since these organs do not appear to have substantial amounts of ferritin (for review see ref. [10]).
6) Intracellular hydrogen ion balance and metabolic removal of H.sup.+ : Applicant also proposes that pyruvate can also influence favorably the cellular hydrogen ion balance. Therapeutically applied pyruvate stimulates hydrogen ion removal by metabolic consumption as opposed to direct chemical buffering or neutralization as meditated by, e.g. bicarbonate or other cellular buffers; thus pyruvate can enhance metabolic removal (cause covalent sequestration of) intracellular hydrogen ions. Virtually all major mammalian cell types have at least four major enzymatic mechanisms at their disposal by which pyruvate metabolism contributes to this type of metabolic consumption of H.sup.+ : a) during reduction of pyruvate to lactate via lactate dehydrogenase one H.sup.+ ion is consumed to form the lactate anion; lactate anion can then be washed out (transported across the cell membrane) by a specific process which comprises obligatory co-export with another H.sup.+ ion (H.sup.+ -symport); this export system is the powerful monocarboxylate transport system of the cell membrane; b) during oxidative decarboxylation of pyruvate by pyruvate dehydrogenase, prior mitochondrial H.sup.+ -import removes cytoplasmic H.sup.+, i.e. for one pyruvate decarboxylated one H.sup.+ ion is stoichiometrically consumed turning up ultimately in the water generated by the mitochondrial respiratory chain; c) during CO.sub.2 -fixation by maleate dehydrogenase (malic enzyme, decarboxylating NADP.sup.+ -dependent malate dehydrogenase) malate anion is formed and again one H.sup.+ ion is stoichiometrically consumed to form malate and NADP.sup.+, d) Furthering the oxidative metabolism of pyruvate plus one H.sup.+ is also the already mentioned anaplerotic pyruvate carboxylase of the mitochondria. Thus, the pyruvate anion functions as natural hydrogen ion remover, gently alkalinizing cells and blood without depending on external buffers like bicarbonate. Thus the applicant contends that pyruvate can possibly function as an alternative to traditional and perhaps less gentle treatments of metabolic acidosis by bicarbonate. The advantage of pyruvate cellular alkalization would be that systemic pyruvate application drives Hydrogen ions out of the cells, whereas bicarbonate drives hydrogen ion into the cells (exacerbating intracellular acidosis) until the excess CO.sub.2 is eliminated via the lungs.
A moderate alkalization is desirable for all mammalian cells or organs that recover from damage associated with intracellular acidification; this would apply to situations where the residual metabolism has become acidotic and must be restarted to reestablish normal ion homeostasis simultaneous with replenishment of crucial cellular metabolite pools (especially that of mitochondrial oxaloacetate) and energy stores (phosphorylation potential). The potential for reductive release of hazardous free iron from cellular complexes (ferritin, myoglobin, cytochromes) will also be diminished by pyruvate, since Fe2+(ferrous Ion) release under intracellular conditions requires the above described accumulation of reducing equivalents in combination with H.sup.+ (acidosis). Clearly, applicant can claim that pyruvate has the potential to influence favorably cellular redox and hydrogen ion balances, via its effects on the cytoplasmic [NAD*]/[NADH]*[H.sup.+ ] ration and its H.sup.+ -consuming metabolic pathways; these features appear to be particularly efficacious in states of partial and reversible cell damage and/or recovery from damage or from extreme stress: reoxigenation after hypoxia, reperfusion after ischemia and myocardial infarct, reestablishing coronary circulation after cardiopulmonary bypass, reperfusion after percutaneous transluminal coronary angioplasty, reperfusion after enzymatic recanalization of thrombotic vessels (streptokinase-type interventions), recovery from excessive catecholamine stress or physical exertion, recovery from probably all types of circulatory shock, if they were associated with hypoxia/ischemia and acidosis.
7) Protection of essential -SH groups: If during cellular damage reductive release of free Fe2+(ferrous Ion) occurred, triggering Fenton-type reactions to produce free hydroxyl radicals, this oxidative stress could still be limited by exploitation of features of pyruvate other than those already discussed. The mechanism is another feature that applicant contends is unique to pyruvate. It has been recognized that the cytotoxicity of oxygen-derived free radicals includes oxidation of labile -SH groups. Optimum functioning of vital enzymes such as Na.sup.+ /K.sup.+ -ATPase and glyceraldehyde-3-phosphate dehydrogenase or metabolite transporters (e.g., the specific mitochondrial pyruvate transporter) appears to depend on such labile -SH groups; other effects of the free radicals include the relatively unspecific peroxidation of membrane lipids which is thought to disturb normal membrane function; possibly, free radicals can also oxidize protein-thiols thus causing direct damage to structural proteins of the cytoskeletal apparatus [17]which can jeopardize the physical integrity and sturdiness of the cell.
Applicant proposes that pyruvate has the potential to strengthen the intrinsic cellular tolerance against this type of oxidative stress on labile but essential -SH groups. This latter mechanism probably operates via the well-known pyruvate-induced citrate accumulation; citrate is an allosteric inhibitor of phosphofructokinase, the main enzyme regulating glycolytic flux. Inhibition of phosphofructokinase leads to an accumulation of glucose-6-phosphate (G-6-P), the immediate phosphorylation product of glucose (hexokinase). G-6-P is also the substrate of the G-6-P dehydrogenase, the first and rate-limiting enzyme controlling the metabolic throughput of the pentose phosphate cycle; it has been shown that increased levels of G-6-P increase the rate of the pentose phosphate pathway in the heart [12]. This metabolic pathway produces reducing equivalents in the form of NADPH.sub.2 which are normally used for reductive syntheses and, importantly, also to keep the glutathione system in its physiologic reduced state. The glutathione system is considered the main cellular defense against sudden oxidative stress due to oxygen-derived and possibly other free radicals. Thus, applicant points out that pyruvate, via an allosteric effect on glycolysis at the level of phosphofructokinase, has the potential to strengthen the reductive capacity of the glutathione system, which will likely improve cellular tolerance to acute oxidative stress. Of significance in this contest is that reduced glutathione (GSH) likely prevents/minimizes oxidation of labile protein-SH groups; maintenance of the cellular GSH/GSSG redox status is therefore likely important for maintaining protein-thiols and enzyme-thiols in their physiologic reduced state. Several powerful enzymes that are instrumental for normal cell function contain labile -SH groups; known examples are the Ca.sup.++ -ATPase of the sarcoplasmic reticulum (excitation-contraction coupling in heart, skeletal and smooth muscle), the creatine kinase (maintaining the cytoplasmic phosphorylation potential in heart, skeletal and smooth muscle and brain), glycogen phosphorylase (mobilization of liver and kidney glycogen to stabilize blood glucose levels), and the already mentioned Na+/K+-ATPase (ubiquitous cellular Na.sup.+ /K.sup.+ homeostasis, affecting also that of calcium via the Na.sup.+ Ca.sup.2++ -and Na.sup.+ /H.sup.+ -exchangers) (for review see ref. [11]). Disabling these enzymes by oxidizing their labile -SH groups is incompatible with long-term cellular survival, not to mention the maintenance of their vital cell- and organ specific functions.
8) Non-enzymatic interaction of pyruvate with hydrogen peroxide: Another novel feature of pyruvate that applicant considers is its capacity to directly neutralize hydrogen peroxide on a 1 to 1 moral basis. Under cellular conditions this interaction is spontaneous and does not require enzyme catalysis; it is an interaction between pyruvate's carbonyl group (alpha-keto group) and hydrogen peroxide yielding carbon dioxide and acetate. This reaction is probably enhanced by the presence of free Fe2+(ferrous Ion). The released carbon dioxide is highly diffusible across all cellular membranes and can thus immediately be washed out from cells, organs or removed from the body via the lung; acetate, the other product of the pyruvate-hydrogen peroxide interaction, can be readily activated by mitochondrial acyl-coenzyme A synthases yielding acetyl-CoA, the main substrate for the citric acid cycle. Applicant points out that this non-enzymatic mechanism of H.sub.2 O.sub.2 -pyruvate interaction could conceivably mitigate the sudden oxidative stress experienced by cells/organs previously compromised by lack of oxygen, lack of circulation, metabolic acidosis, iron overload, extreme metabolic stress.
9) Effect on blood oxygen transport: Pyruvate has also advantageous effects on blood, erythrocytes and their capacity to release oxygen at the specific oxygen tension in the microcirculation. It is well established that pyruvate can increase erythrocyte 2,3-diphosphoglycerate (2,3-DPG) levels [13]; 2,3-DPG is important for the position of the oxygen dissociation curve and hence the release of oxygen from hemoglobin at the partial pressures of oxygen prevailing in the microcirculation. This effect of pyruvate will likely improve the oxygen supply to parenchymal cells; the effect may become especially important in situations where oxygen supply is precarious. This effect of pyruvate requires the presence of adenine or inosine as well as relatively high concentrations of phosphate as additional substrates. The feature has been exploited in blood banking for decades, but applicant points out that this is an additional important argument for novel systemic pyruvate applications in conditions of oxygen deficiencies such as restriction of circulation (ischemia), high altitude (hypoxia), hemodilution (severe external or internal blood loss), severe anemia. However, under high altitude conditions, respiratory alkalosis may develop; if such a conditions exists, pyruvate application may not be justified, since it could aggravate the alkalosis, an effect that may offset the beneficial effect of 2,3 -DPG accumulation on the oxygen dissociation curve.